Nucleic acid probes have a promising future as aids to the diagnosis of infectious and genetic diseases. In order to detect hybridization of the probe to the sample nucleic acid, one may "label" the probe. While radiolabeling is common, the probe may be non-radioisotopically labeled with a ligand, fluorescer, chemiluminescer, enzyme, or antibody. Falkow, U.S. Pat. No. 4,358,535. Use of a nonradioactive label is preferable because of the health hazards, expense and limited half life of radioisotopes.
The term "conjugating agent" is used here to refer to an agent which directly or indirectly, covalently or noncovalently, and reversibly or irreversibly, achieves a binding or association between two chemical species, such as a proteinaceous label and a nucleic acid.
A large number of covalent conjugating agents (crosslinking agents) are known in the immunoassay art, where they are used to label ligands and antibodies. (The same agents are also often used to attach immunogens to carriers.) See, for example, Halmann, U.S. Pat. No. 4,302,534, at column 2, lines 27-57; Parikh, U.S. Pat. No. 4,298,685, column 3, lines 6, 56; Singh U.S. Pat. No. 4,241,177; Rubenstein, U.S. Pat. No. 3,817,837; Farina, U.S. Pat. No. 4,378,428, column 26, line 59 through column 27, line 29; Devlin, U.S. Pat. No. 3,951,748, column 4, line 45 through column 5, line 19; and Albarella, U.S. Pat. No. 4,469,797.
A number of methods are known for covalently crosslinking proteins to nucleic acids. See EP Appl 151,001. Borel, et al., J. Immunol Methods, 67: 289-302 (1984) expressed a strong preference for glutaraldehyde as a crosslinking agent. They used it to bind an oligonucleotide to a protein carrier. Renz, EMBO J., 2: 817 (1983), used the well known cross linker glutaraldehyde to attach histones to nucleic acids. Renz also used benzoquinone to attach a uniformly positively charged polymer to a protein, thus modifying the charge on that protein so it would have sufficient affinity for nucleic acid to be crosslinked by glutaraldehyde.
This method required (1) use of unstable and toxic reagents (benzoquinone) to modify the protein; (2) separation of activated complex from free benzoquinone; (3) attachment of polymer to the activated proteins; and (4) separation of protein-bound polymer from free polymer before conjugation to nucleic acid. In practice, these steps are time consuming and may be difficult to execute properly.
This invention concerns the use of molecules, particularly charged molecules, having positive regions to bind nucleic acids and hydrophobic regions to bind proteins to modify the charge on a protein such that the protein binds the nucleic acid sufficiently to be conjugated in a single step.
It is already known in the art that a wide variety of cationic detergents, possessing hydrophobic groups on one end and positively charged groups on the other, may be prepared. See, e.g., Ohbu, U.S. Pat. No. 4,235,759; Lar, U.S. Pat. No. 4,454,060. The art fails, however, to teach that these detergents may have any value in the conjugation of nucleic acids with proteins. Indeed, cleanser compositions have been devised which are intended to break natural crosslinks in proteins. Schafer-Burkhara, U.S. Pat. No. 4,311,618.
Detergents have been utilized in the immunoassay art, but not in the manner addressed by the present invention. Albert, U.S. Pat. No. 4,486,534 teaches the isolation of immunologically active conjugates from a mixture of active and inactive conjugates by putting the mixture in contact with a carrier-bound complex-former such as a fatty acid, and eluting the inactive conjugates with a detergent. Caldwell, U.S. Pat. No. 4,427,782 teaches the use of an anionic detergent to solubilize an antigen for later purification, and use in the isolation and purification of a corresponding antibody.
Weltman, U.S. Pat. No. 4,002,532 describe use of organic polyamine conditioners in connection with the coupling of enzymes to antibodies. There is no teaching that these conditioners have any value in coupling enzymes to nucleic acids. In any event, the recited polyamines were not cationic detergents.